Tumor Necrosis Factor alpha (TNF-alpha) is a member of a group of cytokines that stimulate the acute phase reaction, and thus is a cytokine involved in systemic inflammation. TNF-alpha is able to induce inflammation, induce apoptotic cell death, and to inhibit tumorgenesis and viral replication. Dysregulation of TNF-alpha production has been implicated in a variety of human diseases like autoimmune disease, ankylosing spondylitis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, Wegener's disease (granulomatosis), Crohn's disease or inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), Hepatitis C, endometriosis, asthma, cachexia, atopic dermatitis, Alzheimer as well as cancer.
Its receptor molecules include TNFR1 and TNFR2. TNF-R1 is expressed in most tissues, whereas TNF-R2 is found only in cells of the immune system. Upon contact with TNF-alpha homotrimers, TNF receptors form trimers and thereby initiate intracellular cell signaling.
Accordingly, soluble TNFR molecules or fragments thereof, which are able to bind to TNF-alpha, can be used as a competitive inhibitor for TNF-alpha. The present disclosure relates to such soluble TNFR2 molecules fused to an Fc portion of a human immunoglobulin (TNFR2:Fc), and more particularly to methods for determining, obtaining and purifying such TNFR2:Fc molecules.
TNFR2:Fc can be manufactured by a bioprocess using recombinant CHO cells, e.g. using dihydrofolate reductase deficient (dhfr-) CHO cells. One particular form of TNFR2:Fc is etanercept which consists of 934 amino acids with an apparent molecular weight of 125 kDa. It comprises a homodimer of the extracellular ligand-binding portion of human tumor necrosis factor receptor (p75) linked to the Fc portion of a human IgG1. The Fc component in both molecules of the homodimer contains the complete hinge, CH2 and CH3 regions, but not the CH1 region of IgG1 (cf. FIG. 1). It is preferably synthesized as a dimeric, secreted, soluble protein while dimerization of the Fc region via three disulphide bonds occurs post-translationally.
By use of X-ray crystallography as well as of mass spectrometry, the complete disulphide bridging pattern of a preferred form of human TNFR2:Fc, etanercept, could be elucidated (see Table 1). Relevant parts of the resolved structures of TNFR2 and its interface with TNF-alpha are shown in FIG. 2 and FIG. 3, whereas connectivity of disulphide variants for the main TNFR2:Fc variant is summarized in Table 1 (see also FIG. 4).
TABLE 1Disulphide bridging pattern of etanerceptIntra-chain (Receptor/Fc-part)Inter-chainCys(18)-Cys(31) Cys(98)-Cys(115)Cys(240)-Cys(240′)Cys(32)-Cys(45)Cys(121)-Cys(139)Cys(246)-Cys(246′)Cys(35)-Cys(53)Cys(142)-Cys(157)Cys(249)-Cys(249′)Cys(56)-Cys(71)Cys(163)-Cys(178)Cys(74)-Cys(88)Cys(281)-Cys(341)Cys(78)-Cys(96)Cys(387)-Cys(445)Cys(104)-Cys(112)
However, misfolded TNFR2:Fc has been found in all analysed TNFR2:Fc preparations. Such misfolded TNFR2:Fc is not preferred when TNFR2:Fc is used in any of the above-noted therapies. U.S. Pat. No. 7,294,481 reports that such misfolded TNFR:Fc such as TNFR2:Fc is formed early in the cell culture process, is transported and represents a significant proportion (about 25-50%) of the expression product. It is further reported that such misfolded TNFR:Fc can be reduced, if the TNFR:Fc producing host cell is cultured at a temperature of 25-34° C. during the production phase. Moreover, it is reported that such misfolded TNFR:Fc can be separated by hydrophobic interaction chromatography. However, as shown in the examples section herein, the currently available TNFR2:Fc preparations (marketed as ENBREL®) still contain wrongly disulphide bridged TNFR2:Fc (see Table 4 below). This may be due to the difficulty of separating same from correctly folded TNFR2:Fc.
Accordingly, there is a need in the art for methods for determining the purity of TNFR2:Fc in a sample—here the amount of wrongly disulphide bridged TNFR2:Fc—which allow for the selection of, e.g., fractions having the desired higher degree of purity.